Intrinsically disordered regions in nucleosome positioning barriers

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Nucleosome depleted promoter regions (NDRs) followed by regular nucleosomal arrays (stereotypic NDR-array patterns) are the universal chromatin pattern of active genes in eukaryotes and are essential for transcription regulation, but remain unexplained. General regulatory factors (GRFs), like yeast Abf1/Reb1 or maybe mammalian CTCF, are not classical transactivators of transcription but suggested to generate NDRs and function as nucleosome barriers for array formation. Indeed, we showed that basic NDR-array patterns are reconstituted in a minimal system of Abf1/Reb1 and ATP dependent chromatin remodelers, like ISW2. Now, we ask what makes a GRF a nucleosome barrier. Besides DNA binding domains, GRFs are highly enriched in intrinsically disordered regions (IDRs). We hypothesize that specific IDR properties are key to the GRF nucleosome barrier function. To study this, we collaborate with the group of Prof. Rohit Pappu (Washington University, St. Louis) that has extensive experience in the biophysics of IDRs and in rational IDR design to interrogate function. First sequence analyses in the Pappu group already showed a characteristic and conserved IDR organization of GRFs, i.e., long acidic IDRs flanking the DNA binding domain, that are distinct from IDR organizations in classical transactivators. This provides a conceptional framework to study which IDR properties confer nucleosome barrier function by a) random mutagenesis (genetic screens); b) domain swaps; c) comparative genomics; and d) de novo design according to IDR biophysics. We will test for barrier function both in vivo as well as by direct biochemical assessment in our unique reconstitution system. This way we will define the specific molecular requirements for nucleosome barrier function.